Dynamic coupling between conformations and nucleotide states in DNA gyrase

Abstract

Gyrase is an essential bacterial molecular motor that supercoils DNA using a conformational cycle in which chiral wrapping of > 100 base pairs confers directionality on topoisomerization. To understand the mechanism of this nucleoprotein machine, global structural transitions must be mapped onto the nucleotide cycle of ATP binding, hydrolysis and product release. Here we investigate coupling mechanisms using single-molecule tracking of DNA rotation and contraction during Escherichia coli gyrase activity under varying nucleotide conditions. We find that ADP must be exchanged for ATP to drive the rate-limiting remodeling transition that generates the chiral wrap. ATP hydrolysis accelerates subsequent duplex strand passage and is required for resetting the enzyme and recapturing transiently released DNA. Our measurements suggest how gyrase coordinates DNA rearrangements with the dynamics of its ATP-driven protein gate, how the motor minimizes futile cycles of ATP hydrolysis and how gyrase may respond to changing cellular energy levels to link gene expression with metabolism.

Figure 1

Gyrase mechanochemical cycle and single-molecule rotor bead tracking assay. (a) Diagram of the gyrase tetramer showing approximate relationships between protein domains, block diagram of gyrase subunits, and schematic of the supercoiling reaction cycle as described in the text. (b) Schematic of the RBT assay. A DNA template containing a strong gyrase site (SGS) is stretched using magnetic tweezers. Angle and z are measured by imaging the rotor from below using evanescent scattering. (c) Plot of cumulative rotor angle and z, showing processive supercoiling during a single gyrase binding event (1 mM ATP, 0.8 pN tension). (d–f) State transitions between Ω, α, and ν are detected on the basis of fits to the angle trace (black line) and application of a threshold to the z trace (dashed horizontal line). (d) 75 μM ATP, 0.8 pN tension; (e) no nucleotide, 0.8 pN tension; (f) 2 mM ATP, 1.4 pN tension. Dwells in the ν state are scored when z is contracted by less than 15 nm. A total of 1415 gyrase encounters such as those shown in c–f were analyzed for this study; see Supplementary Table 4 for detailed statistics.

Figure 2

Conformations of the DNA:gyrase complex probed using RBT. (a–d) Distributions of DNA:gyrase conformations under varying nucleotide and tension conditions. Gyrase binding events, together with flanking data reflecting free DNA (unless otherwise indicated), were excised from angle and z traces and pooled to construct histograms of paired (angle, z) values. See also Supplementary Table 1. The number of gyrase encounters included in each histogram is (a) N=38 (0 ATP, 0.8 pN), N=19 (0 ATP, 1.4 pN); (b) N=20 (75 μM ATP, 0.8 pN), N=48 (75 μM ATP, 1.4 pN); (c) N=9 (1 mM ATP, 0.8 pN), N=74 (2 mM ATP, 1.4 pN); (d) N=26 (2 mM ADP), N=16 (2 mM AMPPNP). (e) Schematic diagram showing conformations of the nucleoprotein complex in the angle-z plane, together with state transitions (arrows) characterized in this study.

Figure 3

Structural dynamics of DNA gyrase associated with ADP/ATP exchange. All data were collected under 0.8 pN tension. (a) Excised regions from traces of cumulative rotations as a function of time with varying [ATP]. Even rotations are indicated by dotted lines, in phase with the equilibrium value of angle prior to enzyme binding. Inset: after fitting angular traces to detect stepwise changes in equilibrium angle, τ₀ was defined as the duration of the major dwell at the ~0 rotation mark. (b) Angular traces acquired in 1 mM ATP with varying [ADP]. See Supplementary Table 4 for the numbers of such angular traces N_gyrase analyzed for each condition in this study. (c) [ATP] and [ADP] dependence of <τ₀>⁻¹. Data are plotted as the inverse of the mean lifetime at the ~0 rotation dwell ± s.e.m. (see also Supplementary Fig. 2). See Supplementary Table 4 for the number of dwells N₀ averaged for each condition. Solid lines are fits to a mechanochemical model (d) in which the Ω state is modeled with all possible combinations of ATP and ADP binding in equilibrium to the two nucleotide binding pockets (see also Methods). The dissociation rate k_off from all Ω states is omitted from the diagram for simplicity. Reversible nucleotide binding transitions are modeled with single-site ATP and ADP affinities K_ATP and K_ADP respectively; overall dissociation constants for binding transitions, incorporating site statistics due the presence of two independent binding sites, are indicated. Pink arrows connect sets of interconverting states grouped within pink dashed lines. Parameter values are quoted in Table 1. (e) Dependence of supercoiling velocity on the [ADP]/[ATP] ratio. Data are coplotted for single-molecule RBT experiments in which either [ATP] (black) or [ADP] (red) was fixed, and bulk malachite green assays where [ADP] was fixed (cyan, see Supplementary Fig. 3). The solid lines are predictions of the model in panel (d). Data are plotted as mean ± s.e.m. See Supplementary Table 4 for the number of cycle times N_cycle averaged for each single-molecule condition. For bulk experiments, three independent experiments were averaged for each data point.

Figure 4

Structural dynamics of DNA gyrase associated with ATP hydrolysis. (a) Excised regions from traces of cumulative rotations as a function of time in the presence of indicated [ATP] and 300 μM AMPPNP, illustrating long dwells in the chirally wrapped α state (arrows). (b) [ATP]-dependence of the rate of exit from α states (<τ_α>⁻¹) in absence of AMPPNP (red) and in presence of 300 μM AMPPNP (black). <τ_α> is related to measurable quantities by <τ_α> = <τ₁-δ>, where τ₁ is the duration of the ~1 or ~1.7 rotation dwell, and δ is the detection threshold. Dwells at ~1 and ~1.7 rotations are scored by automated analysis only if they exceed a detection threshold of 1 s or 4 s respectively, allowing discrimination from adjacent major dwells. The abscissa of the horizontal black line is <τ_α>⁻¹ averaged over all [ATP] conditions. Data are plotted as inverse of the mean lifetime in the α state ± s.e.m. See also Supplementary Fig. 5. (c) Two categories of on-pathway α states can be observed – those followed by completion of a single cycle and dissociation from the subsequent angular plateau, and those followed by completion of the cycle and further Ω-to-α transitions. (d) [ATP]-dependent probability that a scored on-pathway α state is followed by termination of the processive run (solid lines). Dashed lines represent the basal probability that any cycle is followed by dissociation, and is identical to 1-P_T, where P_T is the transition processivity (see Methods and Supplementary Fig. 4). Probabilities are plotted ± standard error $\sqrt{\frac{p(1-p)}{n}}$. (e) Branched mechanochemical model. After formation of an α state with 2 ATP bound, hydrolysis can accelerate strand passage. In the absence of ATP hydrolysis, the enzyme can still access a slower pathway for strand passage, after which it is incapable of processively beginning another cycle. See Supplementary Table 4 for detailed statistics of the number of gyrase encounters N_gyrase analyzed for each condition in presence of 300 mM AMPPNP (panels a, c), the number of α dwells N_α averaged for each lifetime measurement (panel b), and the number of on-pathway α dwells N_α,on used for each probability measurement (panel d).

Figure 5

Structural dynamics of DNA gyrase affected by tension, and [ATP]-dependent analysis of the ν state. (a) Excised angular traces under 1.4 pN tension. (b) Angle and z measured under varying [ATP] and 1.4 pN tension. (c) [ATP] dependence of τ_ν⁻¹, τ₀⁻¹, and the overall supercoiling velocity, comparing 1.4 pN and 0.8 pN conditions (see also Supplementary Fig. 5). ATPase rates measured using a bulk NADH-coupled assay (Supplementary Fig. 3) are coplotted with supercoiling velocity data. Data are plotted as inverse of the mean dwell time ± s.e.m. (left), mean velocity ± s.e.m. (right, bulk measurements) or inverse of the mean cycle time ± s.e.m. (right, single-molecule measurements). Solid lines are fits to a mechanochemical model (d) incorporating the ν state. Reversible ATP binding transitions are modeled with single-site affinity K_ATP. Some reverse transitions are ignored because of fast forward progress. All kinetic parameters other than K_ATP and k₃ have been allowed to vary from values determined at 0.8 pN, and have been determined using measurements of processivities, state lifetimes, and branching ratios (see Methods). Parameter values are quoted in Table 1. See Supplementary Table 4 for the number of gyrase encounters (panels a,b) analyzed for each condition, and for the numbers of ν dwells, ~0 rotation dwells, and cycle times averaged for each lifetime or supercoiling velocity measurement (panel c). For the bulk coupled assay, three independent measurements were made at each [ATP] (see Supplementary Fig. 3 for a plot of individual observations).